CN105779659A - Primer and kit used for detecting foot-and-mouth disease virus type O as well as using method and application of kit - Google Patents
Primer and kit used for detecting foot-and-mouth disease virus type O as well as using method and application of kit Download PDFInfo
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Abstract
The invention discloses a primer and kit used for detecting the foot-and-mouth disease virus type O as well as a using method and application of the kit. The primer is a primer pair FMDO, wherein the forward primer FMDOF is 5'-TGGCCTCAAAGACTCTCG-3' and the reverse primer FMDOR is 5'-CAAGCTACAGATCACTTTAC-3'. The using method of the kit comprises collection of fresh samples of sick animals, nucleic acid extraction, reverse transcription-polymerase chain reaction and detection by agarose gel electrophoresis. The method has strong specificity and high sensitivity, is simple and convenient to operate, saves labor and time and has the effect of improving the accuracy and specificity of detection. The kit can be used for rapidly and conveniently detecting the foot-and-mouth disease type O strains, can be used for epidemiological survey and analysis of the foot-and-mouth disease virus and has a broad market application prospect and higher economic benefits.
Description
Technical field
The present invention relates to a kind of for detect the primer of foot and mouth disease virus O type, test kit, using method and
Application, belongs to biological technical field.
Background technology
Foot and mouth disease (aphthae epizooticae;Foot and mouth disease, FMD) it is by foot and mouth disease virus
Acute, hot, the high degree in contact sexually transmitted disease of a kind of Zoonosis caused, it faces, and to examine feature be in oral cavity
Mucosa, extremity lower end and breast etc. skin forms vesicle and speck.This disease is propagated rapidly, popular wide, becomes
Year, animal took optimum process more, and young animal is many, and because of myocardial damage, mortality rate is higher.Foot and mouth disease is widely current
In all over the world, cause huge economic loss, national governments and international organization and pay much attention to, be listed in
World community decides through consultation the No.1 Notifiable disease put out jointly.Along with the increase increasingly of international trade, material is handed over
Stream and international association are more and more frequent, and prevention and control to foot and mouth disease add difficulty, therefore countries in the world
All spend a large amount of human and material resources and financial resources to study, control and eliminate foot and mouth disease.
Foot and mouth disease virus (Foot-and-Mouth disease virus) belongs to the foot and mouth disease in microRNA Viraceae
Tobamovirus, is minimum in RNA viruses one.Foot and mouth disease virus has pleiomorphism, the feature of easily variation.
According to its serological characteristic, 7 serotypes, i.e. A, O, C, SAT1 (South Africa I), SAT2 can be divided into
(South Africa II type), SAT3 (South Africa type III) and Asia 1 type (Type Asia 1).Without handing between each serotype
Fork immunological phenomena.Each serotype comprises again several hypotypes, also only has part between each hypotype of homotype
Cross-immunity.This characteristic of this virus brings many difficulties to the anti-system of foot and mouth disease.
Foot and mouth disease virus content in vesicle liquid, vesicle skin, lymph fluid and the pyrogenic stage blood of infected animal
The highest, next to that each histoorgan, secretions, Excreta, can long-term existence outside toxin expelling, after bringing down a fever
Virus can come across in breast, excrement, urine, tear, saliva and each internal organs.Foot and mouth disease virus environment to external world
Resistance is very strong, resistance to dry.Under field conditions (factors), toxic tissue and the feedstuff of pollution, drinking-water, forage grass,
The contained virus such as fur and soil still has infectivity within a few days or even several weeks.Foot and mouth disease virus is to bronsted lowry acids and bases bronsted lowry
The most especially sensitive, in the buffer of pH3.0 and more than pH9.0, viral infection by pop-off, 2%~4%
Sodium hydroxide, 3%~5% formalin solution, 5% ammonia, 0.2%~0.5% peracetic acid or 5% sodium hypochlorite
The disinfectant good etc. being foot and mouth disease virus.
Foot and mouth disease is the infectious disease that a kind of infectiousness is extremely strong, once occurring often in popularity, in pastoral area, many
Presenting and be very popular, epidemic situation once occurs, and can the flowing of follower spread rapidly, epidemic situation after the regular period
Just die away.Foot and mouth disease can infect many animals, and artiodactylous animals susceptibility is the highest, susceptibility height
Order is followed successively by cattle, milch cow, yak, Babalus bubalis L., pig, sheep, camel.
Preventing and treating for foot and mouth disease there is no specific medicament therapy at present, mainly takes comprehensive measures for prevention and control and suits the medicine to the illness
Therapy.The most basic way is to eliminate infected animal, band poison animal and thorough disinfection, cuts off route of transmission.
Therefore should strengthen quarantine and foot and mouth disease is monitored, in case foot and mouth disease diffusion.
At present, cut open inspection feature according to epidemiology, clinical symptom and case and can make tentatively examining of foot and mouth disease
Disconnected, but make a definite diagnosis and need to carry out laboratory diagnosis.The separation of virus is that laboratory is examined with qualification and serodiagnosis
Disconnected conventional method, but relatively time consuming arduously, the longest.
Summary of the invention
The technical problem to be solved be to provide a kind of primer for detecting foot and mouth disease virus O type,
Test kit, using method and application thereof, use the test kit of the present invention, it is possible to made diagnosis in 2.5 hours,
A kind of effective molecular biological testing is provided for foot and mouth disease clinical diagnosis and Molecule Epidemiology Investigation.
To achieve these goals, the technical solution adopted in the present invention is: one is used for detecting hoof-and-mouth disease
Poison O type primer, described primer be primer to FMDO, wherein,
Forward primer FMDOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
A kind of test kit for detecting foot and mouth disease virus O type, described test kit include primer to FMDO,
Wherein,
Forward primer FMDOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
Described test kit also includes 5 × RT Buffer, dNTPs, Taq enzyme, RNase inhibitor, reversion
Record enzyme, DEPC water, lysate and flushing liquor.
Described test kit also includes positive control and negative control.
The component of described lysate is: hydroxyquinoline 3~5% (mass fraction), trichloroethyl phosphate
3.2~5.2 μMs, phenyl isothiocyanate 1.5~3.5 μMs, polyvinyl pyrrolidone 1.3~3.5% (mass fraction),
Sodium chloride 0.3~0.5M and sodium citrate 0.6~0.9M.
The component of described flushing liquor is: trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, second
Alcohol 60~80% (volume fraction);pH 7.0.
The using method of a kind of test kit for detecting foot and mouth disease virus O type, comprises the following steps:
(1) collection of sample
(2) extraction of sample RNA
(3)RT-PCR
Sample RNA is carried out RT-PCR amplification, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs
DNTPs 5 μ l, 125 μMs of forward primer FMDOF and 125 μMs of each 2 μ l of downstream primer FMDOR, 25U/ μ l
Taq enzyme 0.5 μ l, 2.5U/ μ l RNase inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l
With 5ng/ μ l RNA 5 μ l;
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 3min;94 DEG C of 40s, 55 DEG C of 45s, 72 DEG C of 30s,
Totally 35 circulations;72℃3min;
(4) detection of RT-PCR amplified production
Pcr amplification product is carried out agarose gel electrophoresis, observed result under gel imaging system.
The collection of described sample is divided into gathering or the collection of liquid animal tissue of solid, shaped animal tissue;
Wherein,
The acquisition method of solid, shaped animal tissue is: aseptic collection animal tissue pathological material of disease 0.1-100mg, loads nothing
RNase pollute centrifuge tube in standby;
The collection of liquid animal tissue is divided into the collection of juice or the collection of blood, the collection side of juice
Method is: gather nasopharynx liquid by Nasopharyngeal swabs, loads in the centrifuge tube without RNase pollution standby;Adopting of blood
Diversity method is: take whole blood, serum or blood plasma 10-100 μ l, adds in the centrifuge tube without RNase pollution standby.
The extraction of described sample RNA, method particularly includes:
(1) animal tissue of solid, shaped first carrying out milled processed, liquid animal tissue is directly used in and carries
Take;
(2) become in animal tissue 20-100mg or the liquid animal tissue 20-100 μ l of pasty state after grinding,
Adding lysate 500 μ l, 12000rpm is centrifuged 30s, isolates supernatant;
(3) supernatant is proceeded in centrifuge tube, adds dehydrated alcohol 500 μ l, forward on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried;
(4) rinsing RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s;
(5) forwarding RNA purification column to another centrifuge tube, add DEPC water 50 μ l, 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
A kind of test kit for detecting foot and mouth disease virus O type answering in terms of detection foot and mouth disease virus O type
With.
Beneficial effects of the present invention
1, nucleic acid extraction process is improved by the present invention, i.e. uses the cracking with available reagent box different formulations
Liquid and flushing liquor, substantially reduce the nucleic acid extraction time, makes the extraction time of nucleic acid by contracting in original 1-2 hour
Short is 2 minutes, saves the time of nucleic acid extraction, thus improves the service efficiency of test kit, makes reagent
The inspection of box is more quick, economical.Wherein, the lysate of the present invention can make sample crack rapidly, release
Go out RNA.Flushing liquor can effectively remove protein, salt and impurity etc., makes extracted RNA purer
Only.
2, the present invention is according to foot and mouth disease virus 3D genome sequence as genes of interest region, is designed to inspection
Survey the specific primer of foot and mouth disease virus O type, other serotypes of foot and mouth disease under equal conditions, can not be expanded, tool
There is the highest specificity.Meanwhile, the test kit of the present invention has the highest sensitivity, and lowest detection is limited to
0.1pg/μl。
3, the invention provides a kind of for detecting foot and mouth disease virus O type test kit, this test kit can quickly,
Detect foot and mouth disease virus O type strain accurately.The single sample of infection of foot-and-mouth disease can be detected, it is possible to right
The detection of mixed infection in clinical case, can be used for the generaI investigation of foot and mouth disease virus cause of disease, Molecule Epidemiology Investigation
And vaccine screens and monitoring.
4, the method for the present invention is built upon on molecular biology mechanism, detection provides negative control and
Positive control, substantially increases the accuracy of detection, reduces false-positive occurrence probability, and specificity is relatively strong,
Time saving and energy saving, the detection time is foreshortened to 2.5 hours by this test kit, substantially increases work efficiency.
5, the invention is particularly suited to the quick diagnosis of a large amount of clinical samples of mouth disease virus infection, Ke Yiwei
In clinic and scientific research, hoof-and-mouth disease preventing and treating and treatment provide scientific basis, and to raising foot and mouth disease virus cause of disease
Detection, monitoring, vaccine development etc. significant.
Accompanying drawing explanation
Fig. 1 is RT-PCR product agarose gel electrophoresis testing result figure;Wherein, swimming lane M represents DL 2000
DNA Marker (is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp) from top to bottom,
Swimming lane 1,2 is foot and mouth disease virus O type negative control sample;Swimming lane 3 is that the foot and mouth disease virus O type positive is right
Product in the same old way, swimming lane 4 is foot and mouth disease virus O pattern product.
Fig. 2 is RT-PCR product agarose gel electrophoresis testing result figure;Wherein, swimming lane M represents DL 2000
DNA Marker (is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp) from top to bottom,
Swimming lane 1 represents foot and mouth disease virus O pattern product;Swimming lane 2-7 represent respectively foot and mouth disease virus A type, c-type,
SAT1 (South Africa I), SAT2 (South Africa II type), SAT3 (South Africa type III) and Asia 1 type (Asia 1
Type) sample.
Detailed description of the invention
Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.
The relative molecular weight of the polyvinyl pyrrolidone used by the present invention is 40000.
Hydroxyquinoline used by the present invention is 2-hydroxyquinoline.
Embodiment 1 is for detecting the test kit of foot and mouth disease virus O type
1, the design of primer
According to the 3D genome sequence of foot and mouth disease virus O type, design primer is to FMDO, wherein,
Forward primer FMDOF:5 '-TGGCCTCAAAGACTCTCG-3 ' (SEQ ID NO.1)
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 ' (SEQ ID NO.2).
2, the extraction of RNA
To make a definite diagnosis the cattle carrying foot and mouth disease virus O type as laboratory animal, extract sample RNA, including following
Step:
(1) collection of sample
Gather cattle whole blood 100 μ l, add in the centrifuge tube without RNase pollution standby.
(2) extraction of sample RNA
A (), in the 100 μ l whole bloods gathered, adds lysate 500 μ l, 12000rpm is centrifuged 30s, isolates
Supernatant;
C supernatant is proceeded in centrifuge tube by (), add dehydrated alcohol 500 μ l, forwards on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried;
D () rinses RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s;
E () forwards RNA purification column to another centrifuge tube, add DEPC water 50 μ l, and 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
The component of lysate is: hydroxyquinoline 3% (mass fraction), trichloroethyl phosphate 3.2 μMs, different sulfur
Phenyl-cyanate 3.5 μMs, polyvinyl pyrrolidone 1.3% (mass fraction), sodium chloride 0.3M and citric acid
Sodium 0.9M.
The component of flushing liquor is: trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol 60%
(volume fraction);pH 7.0.
3、RT-PCR
With sample RNA as template, carry out RT-PCR amplification, simultaneously with swine vesicular disease virus as negative control,
With foot and mouth disease virus O type as positive control, extract negative control and the RNA of positive control sample, RT-PCR
Amplification, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs of dNTPs 5 μ l, 125 μMs of forward primer
FMDOF and 125 μMs of each 2 μ l of downstream primer FMDOR, 25U/ μ l Taq enzyme 0.5 μ l, 2.5U/ μ l RNA
Enzyme inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l and 5ng/ μ l RNA 5 μ l.
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 3min;94 DEG C of 40s, 55 DEG C of 45s, 72 DEG C of 30s,
Totally 35 circulations;72℃3min.
4, the detection of RT-PCR amplified production
Take RT-PCR amplified production 5 μ l and Loading Buffer 5 μ l mixing, be added to 1.0% containing EB
In agarose gel, it is simultaneously introduced negative control, the RT-PCR amplified production of positive control and DL2000
DNA Marker, voltage is 120V, electrophoresis 35min, then observed result (result in gel imaging instrument
See Fig. 1).
Embodiment 2 is for detecting the test kit of foot and mouth disease virus O type
The test kit of embodiment 2 is essentially identical with the test kit of embodiment 1, and difference is: this enforcement
The component of the lysate of example is hydroxyquinoline 5% (mass fraction), trichloroethyl phosphate 4 μMs, isothiocyanic acid
Phenyl ester 2.5 μMs, polyvinyl pyrrolidone 2% (mass fraction), sodium chloride 0.5M and sodium citrate 0.6M.
The component of flushing liquor is trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol 80% (body
Fraction);pH 7.0.
Embodiment 3 is for detecting the test kit of foot and mouth disease virus O type
The test kit of embodiment 3 is essentially identical with the test kit of embodiment 1, and difference is: this enforcement
The component of the lysate of example is hydroxyquinoline 4% (mass fraction), trichloroethyl phosphate 5.2 μMs, different sulfur cyanogen
Acid phenenyl ester 1.5 μMs, polyvinyl pyrrolidone 3.5% (mass fraction), sodium chloride 0.4M and sodium citrate
0.8M.The component of flushing liquor is trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol 70%
(volume fraction);pH 7.0.
The test kit using the embodiment of the present invention 1 extracts the geneome RNA of different sample, to the RNA extracted
Concentration and purity are measured, and result see table.
The RNA concentration of table test kit of the present invention extraction and purity testing result
Sample number into spectrum | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
The RNA concentration extracted | 20.12 | 20.89 | 21.58 | 21.71 | 20.52 | 21.78 | 20.83 |
Absorbance ratio | 1.87 | 1.93 | 1.96 | 1.81 | 1.90 | 1.81 | 1.87 |
Note: RNA concentration unit is ng/ μ l;Absorbance ratio is RNA suction at 260nm and 280nm
Luminosity ratio (260/280), judges the purity of RNA by absorbance ratio, as can be seen from the above table,
Absorbance, between 1.8~2.0, illustrates that the RNA purity extracted is preferable.
In table, sample 1,2,3,4,5,6,7 respectively uses this test kit to extract about from 100 μ l serum
1.00 μ g, 1.04 μ g, 1.08 μ g, 1.09 μ g, 1.03 μ g, 1.09 μ g, 1.04 μ g genomes.
Experimental example
1, for detecting the test kit specificity experiments of foot and mouth disease virus O type
To make a definite diagnosis the cattle carrying foot and mouth disease virus O type as laboratory animal, the checking present invention detects hoof-and-mouth disease
The specificity of the RT-PCR kit of poison O type, comprises the following steps:
(1) collection of sample
Gather cattle whole blood 100 μ l, add in the centrifuge tube without RNase pollution standby.
(2) extraction (formula of lysate and flushing liquor is with embodiment 1) of sample RNA
A (), in the 100 μ l whole bloods gathered, adds lysate 500 μ l, 12000rpm is centrifuged 30s, isolates
Supernatant;
C supernatant is proceeded in centrifuge tube by (), add dehydrated alcohol 500 μ l, forwards on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried;
D () rinses RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s;
E () forwards RNA purification column to another centrifuge tube, add DEPC water 50 μ l, and 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
(3)RT-PCR
With sample RNA as template, carry out RT-PCR amplification, simultaneously with A type, c-type, SAT1 (South Africa
I), SAT2 (South Africa II type), SAT3 (South Africa type III) and Asia1 type (Type Asia 1) sample are right
According to, extract RNA, the RT-PCR amplification of control sample, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs
DNTPs 5 μ l, 125 μMs of forward primer FMDOF and 125 μMs of each 2 μ l of downstream primer FMDOR, 25U/ μ l
Taq enzyme 0.5 μ l, 2.5U/ μ l RNase inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l
With 5ng/ μ l RNA 5 μ l.
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 3min;94 DEG C of 40s, 55 DEG C of 45s, 72 DEG C of 30s,
Totally 35 circulations;72℃3min.
(4) detection of RT-PCR amplified production
Take RT-PCR amplified production 5 μ l and Loading Buffer 5 μ l mixing, be added to 1.0% containing EB
In agarose gel, it is simultaneously introduced RT-PCR amplified production and the DL2000DNA Marker of comparison, electricity
Pressure is 120V, electrophoresis 35min, then observed result (result is shown in Fig. 2) in gel imaging instrument.
Agarose gel electrophoresis result shows, the RT-PCR amplified production of inventive samples RNA is at 1350bp
Band occurs, is consistent with expection, it was demonstrated that RT-PCR amplified production of the present invention is foot and mouth disease virus O type purpose
Fragment.Meanwhile, the A type of comparison, c-type, SAT1 (South Africa I), SAT2 (South Africa II type), SAT3
There is not specific band in (South Africa type III) and Asia1 type (Type Asia 1) sample RT-PCR amplification, says
The RT-PCR kit of the bright present invention has good specificity.
2, for detecting the test kit sensitivity experiment of foot and mouth disease virus O type
The RNA of extraction is taken 1 μ g/ μ l make 10 times of serial dilutions, take respectively 1 μ g/ μ l, 100ng/ μ l, 10ng/ μ l,
1ng/ μ l, 100pg/ μ l, 10pg/ μ l, 1pg/ μ l, 0.1pg/ μ l, 0.01pg/ μ l, 0.001pg/ μ l RNA are carried out
RT-PCR one-step method expands, and result shows that the lowest detection of the present invention is limited to 0.1pg/ μ l, and this ratio is commonly
The sensitivity (1pg/ μ l) of RT-PCR two-step method exceed 10 times.
Claims (10)
1. the primer being used for detecting foot and mouth disease virus O type, it is characterised in that described primer is primer
To FMDO, wherein,
Forward primer FMDOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
2. the test kit being used for detecting foot and mouth disease virus O type, it is characterised in that described test kit bag
Include primer to FMDO, wherein,
Forward primer FMDOF:5 '-TGGCCTCAAAGACTCTCG-3 '
Downstream primer FMDOR:5 '-CAAGCTACAGATCACTTTAC-3 '.
Test kit for detecting foot and mouth disease virus O type the most according to claim 2, it is characterised in that
Described test kit also include 5 × RT Buffer, dNTPs, Taq enzyme, RNase inhibitor, reverse transcription,
DEPC water, lysate and flushing liquor.
Test kit for detecting foot and mouth disease virus O type the most according to claim 3, it is characterised in that
Described test kit also includes positive control and negative control.
Test kit for detecting foot and mouth disease virus O type the most according to claim 3, it is characterised in that
The component of described lysate is: hydroxyquinoline 3~5% (mass fraction), trichloroethyl phosphate 3.2~5.2 μMs,
Phenyl isothiocyanate 1.5~3.5 μMs, polyvinyl pyrrolidone 1.3~3.5% (mass fraction), sodium chloride
0.3~0.5M and sodium citrate 0.6~0.9M.
Test kit for detecting foot and mouth disease virus O type the most according to claim 3, it is characterised in that
The component of described flushing liquor is: trishydroxymethylaminomethane 10 μMs, ethylenediaminetetraacetic acid 1 μM, ethanol
60~80% (volume fraction);pH 7.0.
7. a using method for the test kit for detecting foot and mouth disease virus O type as claimed in claim 3,
It is characterized in that, comprise the following steps:
(1) collection of sample
(2) extraction of sample RNA
(3)RT-PCR
Sample RNA is carried out RT-PCR amplification, reaction system 25 μ l:5 × RT Buffer 5 μ l, 12.5 μMs
DNTPs 5 μ l, 125 μMs of forward primer FMDOF and 125 μMs of each 2 μ l of downstream primer FMDOR, 25U/ μ l
Taq enzyme 0.5 μ l, 2.5U/ μ l RNase inhibitor 1 μ l, 2.5U/ μ l reverse transcription 1 μ l, DEPC water 3.5 μ l
With 5ng/ μ l RNA 5 μ l;
RT-PCR amplification program is: 50 DEG C of 30min, 94 DEG C of 3min;94 DEG C of 40s, 55 DEG C of 45s, 72 DEG C of 30s,
Totally 35 circulations;72℃3min;
(4) detection of RT-PCR amplified production
Pcr amplification product is carried out agarose gel electrophoresis, observed result under gel imaging system.
The using method of the test kit for detecting foot and mouth disease virus O type the most according to claim 7,
It is characterized in that, the collection of described sample is divided into the collection of solid, shaped animal tissue or liquid animal tissue
Collection;Wherein,
The acquisition method of solid, shaped animal tissue is: aseptic collection animal tissue pathological material of disease 0.1-100mg, loads nothing
RNase pollute centrifuge tube in standby;
The collection of liquid animal tissue is divided into the collection of juice or the collection of blood, the collection side of juice
Method is: gather nasopharynx liquid by Nasopharyngeal swabs, loads in the centrifuge tube without RNase pollution standby;Adopting of blood
Diversity method is: take whole blood, serum or blood plasma 10-100 μ l, adds in the centrifuge tube without RNase pollution standby.
The using method of the test kit for detecting foot and mouth disease virus O type the most according to claim 8,
It is characterized in that, the extraction of described sample RNA, method particularly includes:
(1) animal tissue of solid, shaped first carrying out milled processed, liquid animal tissue is directly used in and carries
Take;
(2) become in animal tissue 20-100mg or the liquid animal tissue 20-100 μ l of pasty state after grinding,
Adding lysate 500 μ l, 12000rpm is centrifuged 30s, isolates supernatant;
(3) supernatant is proceeded in centrifuge tube, adds dehydrated alcohol 500 μ l, forward on RNA purification column by several times,
12000rpm is centrifuged 30s, the RNA slightly carried;
(4) rinsing RNA purification column with flushing liquor 500 μ l, 12000rpm is centrifuged 30s;
(5) forwarding RNA purification column to another centrifuge tube, add DEPC water 50 μ l, 12000rpm is centrifuged 30s,
Obtain virus and the RNA of tissue gene group, save backup in-20 DEG C.
10. the test kit for detecting foot and mouth disease virus O type as described in any one of claim 2-6
Application in terms of detection foot and mouth disease virus O type.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108588283A (en) * | 2018-05-02 | 2018-09-28 | 刘书勇 | A kind of detection kit and its detection method of O-shaped foot and mouth disease virus |
CN109517806A (en) * | 2018-12-14 | 2019-03-26 | 中国水产科学研究院珠江水产研究所 | A kind of Tilapia mossambica lake Virus strain and its RPA detection primer and method |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102676691A (en) * | 2011-10-31 | 2012-09-19 | 武汉大学 | H1N1, PRRSV and O-FMDV quadruple detection kit |
-
2016
- 2016-05-23 CN CN201610346055.7A patent/CN105779659A/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102676691A (en) * | 2011-10-31 | 2012-09-19 | 武汉大学 | H1N1, PRRSV and O-FMDV quadruple detection kit |
Non-Patent Citations (2)
Title |
---|
曹丽娟等: "O型***病毒3D基因荧光定量PCR方法的建立及应用", 《中国畜牧兽医》 * |
赵丽等: "《现代基因操作技术》", 31 December 2010 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108588283A (en) * | 2018-05-02 | 2018-09-28 | 刘书勇 | A kind of detection kit and its detection method of O-shaped foot and mouth disease virus |
CN109517806A (en) * | 2018-12-14 | 2019-03-26 | 中国水产科学研究院珠江水产研究所 | A kind of Tilapia mossambica lake Virus strain and its RPA detection primer and method |
CN109517806B (en) * | 2018-12-14 | 2022-05-20 | 中国水产科学研究院珠江水产研究所 | Tilapia lake virus virulent strain and RPA detection primer and method thereof |
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